1. Field of the Invention
There is a continuously expanding interest in being able to conjugate, frequently selectively, a compound to another compound which is polyfunctional. Where both compounds are polyfunctional, the problem of conjugation is exacerbated, if one does not wish all of the functional groups to participate in the reaction. Also, the need to functionalize a polyfunctional compound for conjugating to a polyamino compound will frequently require the introduction of protective groups for alcohols and amines, so that the reactive functionality does not polymerize the compound to be conjugated.
One area of particular interest is the conjugation of a wide variety of haptens and antigens to polypeptides (including proteins), particularly where conjugation is to occur at available amino groups. In preparing antibodies for use in competitive protein binding assays, where the analyte of interest is haptenic, it is generally necessary to conjugate the hapten to an antigen, normally a protein. Where the analyte has a plurality of functionalities which can react with the active functionality to be used for conjugating to the polypeptide, it becomes necessary to introduce removable protective groups to prevent polymerization of the analyte. After conjugation, it is usually difficult to efficiently remove the protective groups.
Where the conjugate is to be used for the preparation of antibodies, the resulting antibodies not only recognize the analyte of interest, but the analyte having the protective groups. This may result in substantially reducing the specificity of the antibody composition for the analyte of interest.
One class of competitive protein binding assays involves the use of enzymes as a label. It is necessary to conjugate the analyte of interest to the enzyme. It is desirable that certain reactive site positions on the enzyme be preferentially conjugated as compared to other reactive site positions. A method which would provide the ability to discriminate to even a partial degree is desirable.
In addition, to have an enzyme which has been modified, whereby the same sites will be conjugated to analytes, regardless of the particular analyte, can provide a number of advantages. For example, in one of the assays which employs an enzyme as a label, it is desirable that the enzyme retain a substantial proportion of its initial activity after conjugation, but when antibody or other receptor is bound to the analytes conjugated to the enzyme, the enzymatic activity is substantially reduced. The fewer the analytes necessary to conjugate to the antibody to obtain the desired degree of reduction in enzymatic activity upon the binding of antibody or other receptor to the conjugated analyte, the more sensitive will be the assay response.
In addition, where a universal reagent can be employed for conjugation, greatly increased experience can be obtained in the handling of the compounds, the reacting of the compounds, as well as the subsequent handling and treatment after conjugation. This can provide for great efficiencies in synthesizing and subsequent formulation.
2. Description of the Prior Art
Kato, et al, Eur. J. Biochem. 62, 285 (1976), discloses the use of maleic anhydride with a polyamino compound to provide one or more maleimide groups, followed by the addition of a compound with a mercapto group to add to the double bond of the maleimide. See also, Lee and Kenny, Clinical Chem. 21, 967 (1975).